Project Summary RNA-guided DNA endonucleases derived from bacterial CRISPR systems promise to rapidly advance genome editing-based therapeutics. However, this approach is significantly limited by the ability to target only a mutated allele in the human genome in a safe and specific manner. Huntington's disease (HD) is caused by such a dominant genetic defect and there is clear evidence that mutant allele disruption can positively impact disease progression. We have previously published a safe and efficient method to deliver CRISPR-Cas9 to neurons in vivo and the key limitation we now face to bringing a CRISPR based therapeutic to clinical trials is the lack of therapeutically relevant guides that target the mutant Huntingtin (HTT) gene. To this end, we aim to identify CRISPR guide RNAs that will reduce the burden of mutant HTT protein on the cell. To accomplish this we propose a screen that can rapidly readout the effect a genetic edit has on both copies of the HTT gene in high- throughput and on a single cell basis. We will apply this screen to test thousands of guides for activity and allele specificity. If successful, these experiments will advance a core technology for genome editing and identify individual products that will be rigorously characterized in preclinical work for a HD therapeutic product during an application-oriented Phase II.